This application claims priority to Great Britain Application No. GB 9916757.9 filed Jul. 17, 1999.
The present invention relates to the novel use of the SREBP-cleavage activating protein (SCAP) in a screening method, particularly a method of screening for agents which compete for binding on SCAP, and to agents having SCAP binding characteristics for use in combating diseases associated with elevated lipid levels.
Cholesterol is transported in the blood in the form of protein/lipid complexes termed lipoproteins. Each lipoprotein plays a role in the transport of lipid from peripheral tissues to the liver or vice versa. Prolonged elevation of some of these classes of lipoproteins can lead to deposition of cholesterol and cholesterol esters in the arteries which may in turn lead to arterial occlusion and clinical conditions such as myocardial infarction and heart failure.
Numerous studies have shown a correlation between extent of atherosclerosis and the incidence of myocardial infarction and plasma level of low density lipoprotein (reviewed by Simon A et al., Circulation 96:2449-2452 (1997)). During the past decade, several clinical trials using inhibitors of HMGCoA-reductase, the rate limiting enzyme of cholesterol synthesis, have confirmed the beneficial effect of decreasing plasma LDL levels on cardiovascular mortality (Brown A. S. et al., J. Am. Coll. Cardiol. 32:665-672 (1998). The mechanism by which cholesterol depletion in hepatic cells prevents the down regulation of the LDL receptor, which mediates LDL re-uptake, has been well characterised by Brown and Goldstein (reviewed in Cell 89:331-340 (1997)). They have identified a responsive element in the LDL-receptor promoter which is involved in LDL-receptor regulation by sterols, the Sterol Responsive Element (SRE) (Briggs M. R. et al., J. Biol. Chem. 268:14490-14496 (1993)). They have purified and cloned two SRE binding proteins (SREBPs) present as precursor forms in the endoplasmic reticulum membrane (Yokoyama C. et al., Cell 75:187-197 (1993) and Hua X. et al., Proc. Natl. Acad. Sci. 90:11603-11607 (1993). Upon cholesterol depletion, these membrane bound forms are sequentially cleaved by two recently cloned proteases, S-1-P (Sakai J et al, Mol. Cell, 2:505-514 (1998)), and S-2-P (Rawson R. B. et al, Mol. Cell, 1:47-57 (1997)), and the mature forms thus released, migrate to the nucleus and bind to SRE (Sakai J. et al, Cell 85:1037-1046 (1996). The SREBP-cleavage activating protein (SCAP) has been identified and shown to interact physically with SREBPs (Sakai J. et al., J. Biol. Chem. 272:20213-20221 (1997). The presence of a putative sterol binding domain on SCAP between amino acid 280 to 444 further suggests its role is that of a xe2x80x9ccholesterol sensorxe2x80x9d (Hua X. et al.; Cell 87:415-426 (1996)). Excess cholesterol in the cell, presumably prevents the SCAP/SREBP interaction, thus blocking the SREBP proteolytic maturation process. Cholesterol and its metabolites act as repressors of genes that increase cellular cholesterol content e.g. those catalysing de novo cholesterol synthesis or mediating uptake of cholesterol-rich LDL (Javitt N. B., Faseb 9:1378-1381 (1995)). The water-soluble cholesterol analogue 25OH-cholesterol is a potent suppressor of sterol-regulated genes (Brown M. S. and Goldstein J. L., J. Biol. Chem., 249:7306-7314 (1974)) and has been used to study SCAP function (Nohturfft A. et al., Proc. Natl. Acad. Sci. 93:13709-13714 (1996)).
A first aspect of the invention is a method for screening for a SCAP antagonist for use in combating diseases associated with circulating elevated levels of LDL-cholesterol, said method comprising detecting or assaying the extent or result of transcriptional activity or binding competition on SCAP between a test SCAP antagonist and a control SCAP antagonist, or between a test SCAP antagonist and cholesterol, or a metabolite thereof.
In an alternative or yet further aspect, there is provided a method for the treatment of conditions resulting from elevated circulating levels of LDL-cholesterol and/or triglycerides comprising administration of a SCAP antagonist.